Bioinformatic analysis and experimental validation of the potential gene in the airway inflammation of steroid-resistant asthma

Steroid-resistant asthma is a troublesome clinical problem in public health. The pathogenesis of steroid-resistant asthma is complex and remains to be explored. In our work, the online Gene Expression Omnibus microarray dataset GSE7368 was used to explore differentially expressed genes (DEGs) between steroid-resistant asthma patients and steroid-sensitive asthma patients. Tissue-specific gene expression of DEGs was analyzed using BioGPS. The enrichment analyses were performed using GO, KEGG, and GSEA analysis. The protein–protein interaction network and key gene cluster were constructed using STRING, Cytoscape, MCODE, and Cytohubba. A steroid-resistant neutrophilic asthma mouse model was established using lipopolysaccharide (LPS) and ovalbumin (OVA). An LPS-stimulated J744A.1 macrophage model was prepared to validate the underlying mechanism of the interesting DEG gene using the quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A total of 66 DEGs were identified, most of which were present in the hematologic/immune system. Enrichment analysis displayed that the enriched pathways were the IL-17 signaling pathway, MAPK signal pathway, Toll-like receptor signaling pathway, and so on. DUSP2, as one of the top upregulated DEGs, has not been clearly demonstrated in steroid-resistant asthma. In our study, we observed that the salubrinal administration (DUSP2 inhibitor) reversed neutrophilic airway inflammation and cytokine responses (IL-17A, TNF-α) in a steroid-resistant asthma mouse model. We also found that salubrinal treatment reduced inflammatory cytokines (CXCL10 and IL-1β) in LPS-stimulated J744A.1 macrophages. DUSP2 may be a candidate target for the therapy of steroid-resistant asthma.

Microarray data. The microarray dataset GSE7368, constructed by Goleva et al., was retrieved from the Gene Expression Omnibus (GEO, https:// www. ncbi. nlm. nih. gov/ geo/), which allows researchers to search and download expression data for analysis based on the GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array platform. The experiment contained gene array studies of BALF cells, consisting of 3 steroidresistant (SR) asthma patients and 3 steroid-sensitive (SS) asthma patients. The data were normalized using the gcrma package (version 2.0.0) 13 . All analyses were performed in the R environment (version 3.6.2). The probes that have no expression in most of the samples were removed. The probes were annotated as gene symbols based on the annotation information of the GPL570 platform. An expression value for every gene was acquired based on mean expression estimates. As a result, we obtained the gene expression matrix. Differential expression analysis. Differential expression analysis was performed to identify the DEGs using a limma package (version 3.42.2) 14 . P values were calculated using the t-tests. The DEGs were determined according to the following criteria: (1) a |log2 (fold-change)|> 1 and (2) a P value < 0.05. The volcano plot and the heatmap for the DEGs were constructed using ggplot2 (version 3.3.1) and pheatmap (version 1.0.12) packages 15,16 . We selected the top 6 upregulated DEGs to create a violin plot.
Tissue-specific expressed gene analysis. Online database BioGPS (http:// biogps. org) (version 94eefe6) was used to analyze the tissue-specific expressed DEG genes. The genes identified as highly tissue-specific genes were similar to the previous study described, with more than > 30 multiples of the median (MoM) and less than 1/3 of the highest expressions in the second highest level 17 . Functional enrichment analysis of DEGs. The functional enrichment analysis of DEGs was performed using the clusterProfiler package (version 3.14.3) 18 . The enrichment analyses were performed for predicting Animal experiments. Male C57BL/6 mice (6-8 weeks of age) were used to develop a steroid-resistant neutrophilic asthma (NA) model as described by previous studies with minor modification [23][24][25][26] . All mice were divided into five groups (n = 4 per group) randomly: Control group, NA group, NA + salubrinal group, NA + Dex group, and Control + salubrinal group. Briefly, ovalbumin (OVA, 100 μg) and lipopolysaccharides (LPS, 10 μg) in 50 μl PBS were intratracheally delivered to the mice under anesthesia with subcutaneous pentobarbital on day 0 and day 6. Then the mice were challenged with 5% aerosolized OVA for 40 min on day 13. The mice were sacrificed on day 14, and the lungs were extracted for histology analysis using hematoxylin and eosin (H&E) and Masson staining 27,28 . In some groups, the LPS/OVA-sensitized mice were injected intraperitoneally with dexamethasone (Dex, 1 mg/kg) or salubrinal (2 mg/kg) before the OVA challenge on day 13. The control mice were administered intratracheally with 50 μL PBS, then challenged with PBS for 40 min on day 13. In another group, the control mice were injected intraperitoneally with salubrinal (2 mg/kg) before the PBS challenge on day 13. Quantitative reverse transcription polymerase chain reaction (qRT-PCR). Total RNA was extracted from cells with Trizol (Invitrogen). The cDNA was obtained using the reverse transcription kit (Vazyme). qRT-PCR was conducted according to the instructions of the manufacturer (Vazyme). The primers are displayed in Table 1.
Statistical analysis. All data were expressed as mean ± standard error (SE) and analyzed with SPSS 19.0 statistical software. The student's t-test was performed to calculate the difference between the two groups. The one-way ANOVA followed by Tukey's post hoc analysis was used to calculate the differences in more than two groups. The difference was significant when a P value was < 0.05.

Results
Differentially expressed genes. We analyzed the microarray dataset GSE7368 from the GEO database and selected DEGs between BALF cells from steroid-resistant asthma (SR) and steroid-sensitive (SS) asthma using R packages. Principal-component analysis displayed that the center of the SR group lay far apart from the center of the SS group in space, indicating the different expressions of genes between the two groups (Fig. 1A). A total of 57 upregulated and 9 downregulated genes were determined as DEG genes from SR patients and SS patients, as displayed in Table 2. The volcano plots showed significantly upregulated or downregulated DEGs in SR patients compared with SS patients (Fig. 1B). The red dots indicate the significantly upregulated genes, while the blue dots indicate the significantly downregulated genes. Hierarchical clustering heatmaps manifested the distinguishable DEGs expression pattern between SR patients and SS patients (Fig. 1C). These results above showed that the top 6 DEGs between SR patients and SS patients were IL6, CXCL8, TNF, DUSP2, ADM, and CXCL1.

Functional and pathway enrichment analyses of DEGs.
To explore the function of DEGs between SR patients and SS patients, we performed GO and KEGG enrichment analyses. The biological processes (BPs) of DEGs were mainly enriched in cytokine activity, chemokine activity, and cytokine receptor binding. The cellular components (CCs) of DEGs were mainly involved in the membrane region, membrane microdomain, and membrane draft. The molecular functions (MFs) of DEGs were mainly enriched in the cellular response to lipopolysaccharide, response to the molecule of bacterial region, and response to lipopolysaccharide ( Fig. 2A). The DEGs enriched in GO function enrichment were visualized using the ClusterProfiler package. The yellow dots indicated the GO categories, the color of the line indicated the association between dots and the category, and the size of a dot showed the gene numbers (Fig. 2B). As for the KEGG pathway enrichment analysis, DEGs were enriched in the IL-17 signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, and cytokine-cytokine receptor interaction (Fig. 2C). Our results showed that the functional and pathway enrichment of DEGs was mainly related to cytokine responses.
Gene set enrichment analysis. The GSEA analysis was performed to explore the enriched gene-related pathways. The pathways related to cytokine cytokine receptor interaction, MAPK signal pathway, Toll-like receptor signaling pathway, T cell receptor signaling pathway, natural killer cell mediated cytotoxicity, and hematopoietic cell lineage pathway were mainly enriched in the SR group ( Fig. 3A-F). Our results suggested that the enriched gene-related pathways were mainly associated with cytokine or immune responses.

PPI networks of DEGs between SR patients and SS patients. The protein association networks of
DEGs between SR patients and SS patients were constructed by STRING. A PPI network with 60 nodes and 191 edges had an interaction score > 0.4 visualized by Cytoscape software (Fig. 4A). The nodes indicate genes, and the edges indicate the associations between genes. We used the MCODE plugin in Cytoscape to identify the key PPI network module with the highest score, consisting of 14 genes (Fig. 4B). Furthermore, the top 10 hub genes were identified by the Cytohubba in Cytoscape (Fig. 4C). The functional enrichment analysis showed that these top 10 hub genes were primarily related to the IL-17 signaling pathway, TNF signaling pathway, and NF-kappa B signaling pathways (Fig. 4D). These results revealed that DEGs between SR patients and SS patients were more likely to be related to inflammatory cytokine response.
Validation of DUSP2 in an asthma mouse model. Our analysis indicated that DUSP2 was potentially associated with steroid-resistant asthma. To our knowledge, there are few studies relating to the roles of DUSP2 in steroid-resistant asthma. Therefore, we hypothesized that inhibition of DUSP2 showed a protective effect on the airway inflammation of steroid-resistant asthma. We generated a steroid-resistant NA mouse model and administrated salubrinal according to the experimental procedure shown in Fig. 5A. It was demonstrated that more neutrophil infiltrations in BAL fluids were observed in the steroid-resistant murine asthma model 30 .
Our study suggested that neutrophil numbers were not decreased by Dex treatment in the NA group (LPS/ OVA + Dex) but were reduced after salubrinal administration in the NA group (LPS/OVA + salubrinal) (Fig. 5B). No significant changes in eosinophil, macrophage, and lymphocyte numbers of BAL fluids were observed in the NA group with or without salubrinal treatment (Fig. 5B). Besides, our study revealed that peribronchial inflam- www.nature.com/scientificreports/ mation infiltration and subepithelial collagen deposition were more severe in the NA group compared to the control group, and Dex failed to reverse lung morphological changes based on the inflammation score and Ashcroft score of fibrosis (Fig. 5C,D). As mentioned in the bioinformatic analysis above, DEGs were mainly related to IL-17 and TNF pathways. As a result, our study displayed that the NA group had the highest mRNA levels of IL-17A, TNF-α, and DUSP2 ( Fig. 5E-G). The mRNA levels of IL-17A, TNF-α, and DUSP2 were not reduced by Dex administration in the NA group but were significantly reduced after salubrinal treatment in the NA group ( Fig. 5E-G). These results indicated that the DUSP2 inhibitor ameliorated neutrophilic airway inflammation and cytokine responses (IL-17A and TNF-α)in an asthma mouse model with steroid-resistant.   6A). Furthermore, the elevation of inflammatory cytokines (CXCL10, IL-1β) in LPS-stimulated J774A.1 macrophages was suppressed by salubrinal (Fig. 6B,C). Taken together, our results revealed that the correlation between DUSP2 and inflammatory cytokines (CXCL10, IL-1β) may be involved in the inflammatory response of LPS-stimulated J744A.1 macrophages.

Discussion
Increased morbidity is observed in steroid-resistant asthma patients, and steroid-resistant asthma is more likely to be related to neutrophilic inflammation 5,31,32 . In this respect, it is urgent to explore alternative treatment options for steroid-resistant asthma. Our computational data-mining analysis explored several genes associated with steroid-resistant asthma, aiming to provide new insight into the pathogenesis of steroid-resistant asthma. Different from the original source of the data set, our study pays more attention to the evaluation and correction of data. We use the limma package to search for differential genes, thereby improving the power of the test.
In the current study, we conducted a discovery-driven analysis to identify DEGs in cells of BAL fluids from SR asthma patients and SS asthma patients. 66 DEGs were identified, consisting of 57 upregulated genes and 9 downregulated genes, which were consistent with activated macrophage-related genes. The top six upregulated DEG genes (IL6, CXCL8, TNF, DUSP2, ADM, and CXCL1) in SR asthma patients compared with SS asthma patients were identified. Recent studies have indicated that reduced expression of IL6 suppressed the development of steroid asthma 33,34 . CXCL8, also named IL8, is a CXC chemokine that could recruit neutrophils in steroidresistant airway inflammation 35,36 . Dex could not reduce the levels of IL6, and IL8 in the macrophages of severe asthma patients 37 . It was found that patients with steroid-resistant showed a higher level of TNF-α than patients with steroid-sensitive in C. pneumoniae seropositive asthmatics 38 . Dex could not suppress the levels of TNF-α in steroid-resistant asthma mouse models 32 . LIGHT and LTαβ, which belong to the TNF family, were reported to induce a steroid-resistant inflammatory response in airway epithelial cells 39 . DUSP2, also named phosphatase of activated cells 1 (PAC-1), was reported to function as dephosphorylating threonine and tyrosine residues of targeting substrates 40,41 . DUSP2, predominantly expressed in hematopoietic cells, was demonstrated to upregulate the production of proinflammatory cytokines in immune cells 42,43 . However, few studies have investigated the role of DUSP2 in steroid-resistant asthma. It is necessary to further delineate the pathogenesis role of DUSP2 in steroid-resistant asthma. One study has revealed that plasma ADM was increased in the acute attack period of asthma compared to the remission period of asthma 44 , suggesting ADM may be involved in the process of asthma. CXCL1 was observed to be a kind of neutrophil-related chemokine 45 , which was elevated in neutrophilic   www.nature.com/scientificreports/ asthma patients 46 . Immune cells were reported to play important roles in the pathogenic development of asthma, and asthma is characterized by granulocytic inflammation infiltration in the airways 44,47 . In our study, the tissuespecific expression analysis indicated that the hematologic/immune system was the most highly specific system of the DEGs, suggesting the common occurrence of immune response in steroid-resistant asthma. Enrichment analyses were constructed to explore the roles of DEGs. Results from the GO analysis revealed that the DEGs were mainly enriched in cytokine activity, chemokine activity, cytokine receptor binding, membrane region, membrane microdomain, membrane draft, cellular response to lipopolysaccharide, response to molecule of the bacterial region, and response to lipopolysaccharide. Cytokine-cytokine receptor interaction was reported to be more enriched in neutrophilic asthma than in eosinophil asthma 48 . Lipopolysaccharide (LPS), a component of gram-negative bacteria, was reported to chemoattract neutrophils 49 . These biological pathways are mainly associated with an inflammatory response. KEGG analysis demonstrated that the DEGs were mainly enriched in the IL-17 signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway, NOD-like receptor signaling pathway, and cytokine-cytokine receptor interaction. After analyzing the PPI network of DEGs, we found that the top 10 hub genes identified by the Cytohubba in Cytoscape were primarily enriched in the IL-17 signaling pathway, TNF signaling pathway, and NF-kappa B signaling pathways, which were consistent with the KEGG enrichment analysis. IL-17 signaling pathway has been suggested to mediate the pathogenesis of steroid-resistant asthma, which has the function of neutrophil chemotaxis 50,51 . Nuclear factor-κB (NF-κB) was reported to lead to epithelial cell inflammation of asthma and may be a potential marker for asthma severity 52 . Tumor necrosis factor-alpha (TNF-α) was reported to be associated with steroid-resistant asthma and asthma exacerbation frequency 53,54 . These functional enrichment analyses may exhibit the enrichment of DEGs in a neutrophil-related inflammatory response in steroid-resistant asthma. Different from the previous study 55 , our www.nature.com/scientificreports/ study has added the enrichment analyses of DEG genes, which suggested that neutrophil-related inflammatory responses were implicated in steroid-resistant asthma.
The GSEA data revealed that cytokine cytokine receptor interaction, MAPK signal pathway, Toll-like receptor signaling pathway, T cell receptor signaling pathway, natural killer cell mediated cytotoxicity, and hematopoietic cell lineage pathway were mainly enriched in SR patients. The p38 MAPK pathway activation was reported to be involved in steroid resistance of asthma 56 . Dex failed to inhibit NTHi-induced steroid-resistant allergic airway inflammation, by activating the p38 MAPK pathway 57 . Immune cells were involved in the pathogenic process of asthma 44 . More than half of the pulmonary CD4 + CD3 + cells in moderate-to-severe asthma patients were natural killer T cells, with elevated invariant T-cell receptor expression 58 . Toll-like receptors (TLRs) were reported to be involved in macrophage-related steroid-resistant AHR 59,60 . The modulation of the T-cell receptor (TCR) in CD4 + T cells recruited inflammatory cells and elevated cytokine production in the airway of asthma 61 . Our findings support the association between immune cell activation, inflammatory response, and steroid-resistant asthma.
The DUSP2 gene was reported to be involved in the immune activation process, which could enhance inflammatory responses 43 . However, few studies have reported the role of DUSP2 in steroid-resistant asthma. Since our study suggested that DUSP2 was upregulated in the BAL cells of SR asthma patients as compared with SS asthma patients, we further investigate the role of DUSP2 on steroid-resistant asthma. Previous studies have suggested that salubrinal was a candidate drug for inhibiting the expression of DUSP2 62,63 . Salubrinal is a 480-Da agent that inhibits a serine phosphatase, protein phosphatase 1 (PP1) and elevates the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) 64 . It was suggested that LPS significantly increased DUSP2 expression but caused a decrease in the p-eIF2a/eIF2a ratio, which was reversed by salubrinal in the LPS-induced intraneural hemi-Parkinson disease (PD) model 62 . Through eIF2α, salubrinal could mediate transcriptional and translational regulation 63,65 . Therefore, we can assume that salubrinal may inhibit the transcriptional and translational process of DUSP2 by acting on p-eIF2a/eIF2a ratio. It was found that the administration of salubrinal in mouse models of arthritis significantly reduced inflammatory responses 63 . Interestingly, our findings revealed that administration of a candidate synthetic DUSP2 inhibitor (salubrinal) before the challenge reduced the neutrophilic airway inflammation and cytokine responses (IL-17A, TNF-α) of the steroid-resistant asthma mouse model, suggesting a protective effect of salubrinal in steroid-resistant asthma. Macrophages are considered to be the main immune cells in asthma 66 . Results from a previous study indicated that salubrinal reduced the DUSP2 expressions in both RAW264.7 macrophages and Jurkat cells 63 . In line with the previous studies, our study supports that salubrinal is an inhibitor of DUSP2. It was founded that salubrinal inhibited inflammatory cytokines (IL-1β, IL-6, TNF-α), and DUSP2 expressions stimulated by LPS 62,63 . CXCL10 is a kind of inflammatory cytokine, which enhanced type 1 inflammation resulting in severe pathology 67 . IL-1β was reported to induce neutrophilic inflammation 68 .   www.nature.com/scientificreports/

Limitations
That said, there are some limitations of the present study. First, the sample size of asthma patients in the public datasets was small. More BAL cell expression profiles of asthmatics will be needed in future analysis. Second, more potential in vitro mechanisms should be determined in further study.

Conclusion
In conclusion, several DEGs and enriched pathways in the SR patients compared to the SS patients were identified. Our study not only provides an insight into the role of DUSP2 and its inhibitor (salubrinal) in steroidresistant neutrophilic airway inflammation but also lay a foundation for the development of alternative therapy options in steroid-resistant asthma.   Data were expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, vs LPS group. #P < 0.05, ##P < 0.01, ###P < 0.001, vs control group.